The p53 tumor suppressor is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. It is maintained at low levels in unstressed cells, but stabilized and activated upon DNA damage by means of extensive PTM. Our research has focused on identifying and exploring the roles of p53 PTMs to better understand how they modulate p53 function. Reciprocal negative regulation of p53 and Nanog maintains differentiation. p53 supports the differentiation of embryonic stem cells (ESC) into differentiated states through suppression of NANOG, a gene required for ESC self-renewal. Previously, we showed that p53 Ser315 phosphorylation was important for the suppression of Nanog expression during mouse ESC differentiation in a model containing a chimeric humanized p53 gene. p53 also suppresses dedifferentiation by maintaining suppression of NANOG in differentiated cells, a mechanism of tumor suppression demonstrated to be important in several human cancers, including gliomas and breast cancer. We investigated the roles of induced expression of Nanog in tumorigenesis and metastasis using an engineered mouse model. We demonstrated that co-expression of Nanog and the oncogene Wnt in the mammary tissues of mice promoted tumorigenesis and metastasis. Overexpression of Nanog activated the focal adhesion and calcium signaling pathways and suppressed the p53 signaling pathway, leading to increased tumor cell mobility, invasiveness and metastasis. Analysis of changes in gene expression between control tumors and tumors expressing high levels of Nanog revealed that the promoters of the most highly up-regulated genes exhibited the presence of Nanog transcription factor binding sites as well as the presence of both activating (H3K4me3) and repressive (H3K27me3) histone modifications. These results suggest that expression of Nanog in differentiated cells leads to inappropriate expression of genes with poised promoters that contribute to the metastasis of tumor cells. Global effects of p53 PTM: Mouse models containing missense mutations at p53 PTM sites have been used to investigate the complex effects of p53 PTMs in a physiological setting. Our quantitative mass spectrometry studies of Ser18Ala knock-in mice demonstrated that the mutation affected proteins with roles in energy and metabolism pathways following ionizing radiation. As p53 has been shown to have important roles in regulation of metabolism and energy pathways, we have initiated studies to further understand the modulation of p53-dependent effects on metabolism. As a complement to mouse models, we are using genomic editing techniques for introducing specific modifications into genes in human cells. These methods will allow a more comprehensive investigation of the interrelationships between p53 PTMs and metabolism. Effects of p53 N-terminal phosphorylation on its protein-protein interactions: One of the naturally expressed isoforms of p53, deltaNp53, lacks the first transactivation domain (TAD1) of p53 but does contain the second transactivation domain (TAD2). The expression and stability of the two proteins are affected differently by cell type, cell cycle phase and exposure to various stresses. p53 and deltaNp53 form heterotetramers and the relative abundance of deltaNp53 influences the transactivation activity and target gene specificity of p53. Our characterization of the binding of TAD1 and TAD2 of p53 to the Taz2 domain of the transcriptional coactivator p300 demonstrated that although the two domains bound to Taz2 with equal affinity, the binding of TAD1 was affected by p53 phosphorylation, whereas the binding of TAD2 was unaffected. To better understand the differences between the complexes of Taz2 with TAD1 and TAD2, we have determined the NMR solution structure of a p53 TAD2 peptide in complex with Taz2. Upon binding to Taz2, p53 TAD2 forms a short alpha-helix, similar to the complex-dependent formation of a TAD1 alpha helix in the TAD1-Taz2 complex, but with opposing orientation. The differing orientations allow the conserved phenylalanine and tryptophan residues of the two p53 domains to bind in the same pocket on the surface of Taz2. Concomitant mutagenesis and binding studies have helped further characterize the complex. Comparison of the structures of the two complexes sheds light on how these two similar domains within p53 may function differently in co-activator recruitment after stress and suggests reasons for differences in transactivation between p53 and deltaNp53. In addition, as several new structures of p53 TAD2 complexes have been published, comparison of these structures with the TAD2-Taz2 complex provide new understanding of the importance of flexibility in this domain for the formation of critical protein-protein interactions. Another N-terminally deleted natural isoform of p53, delta133p53, also negatively regulates p53 activity and forestalls the development of cellular senescence. In collaboration with Dr. Curtis Harris, we observed that the age-dependent accumulation of senescent (CD28-CD57+) cells in peripheral blood CD8+ T lymphocytes correlated with decreased delta133p53 expression levels. Functional studies demonstrated that overexpression of delta133p53 restored CD28 expression and cellular proliferation, whereas knockdown of delta133p53 expression induced senescence. These studies suggest that restoration of delta133p53 expression may provide therapeutic benefit in treating immunosenescent disorders, including those associated with aging, cancer, autoimmune diseases and HIV infection. Moreover, also with Dr. Harris we recently demonstrated that, unlike full-length p53, that is subject to proteasomal degradation,delta133p53 is degraded by autophagy during replicative senescence. Thus, delta133p53 also represents a functional and regulatory link between cellular senescence and autophagy, two cellular phenotypes involved in aging and cancer. Functional effects and interplay of p53 C-terminal modifications: The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitinylation, sumoylation, and neddylation, that are primarily localized to the terminal thirty residues of the protein. We are interested in understanding the specific effects of individual site-specific modifications and the interplay between them. We have investigated the effects of mono- and dimethylation of p53 Lys382, a site that alternatively can be methylated, acetylated, or ubiquitinylated. Mono-methylation of p53 Lys382 results in repression of the activity of p53 as a transcription factor and we have continued to investigate the mechanism of repression. Dimethylation of p53 Lys382 is critical for the interaction of p53 with the tandem Tudor domain (TD) of the DNA damage response mediator 53BP1. We have been exploring the role of an additional modification, acetylation at p53 Lys381 within the C-terminal regulatory domain that may combine with Lys382 dimethylation to further modulate the binding of p53 to the TD domain.. Recently, we have shown that the double PTM causes a significant conformational change in the p53 structure when bound to the TD, converting the beta-hairpin of p53K382me2 into an alpha-helix in p53K381acK382me2. Other PTMs, including phosphorylation of serine and threonine residues of p53, further influence the interaction with TD. Our data suggest a novel p53 regulatory mechanism in which different combinations of PTMs enable distinct conformations in p53 when bound to specific interactors.